ALTERED G(S)ALPHA N-TERMINUS AFFECTS G(S) ACTIVITY AND INTERACTION WITH THE G-BETA-GAMMA SUBUNIT COMPLEX IN CELL-MEMBRANES BUT NOT IN SOLUTION

The stimulatory G protein (G(s)) mediates activation of adenylylcyclas e by a ligand-receptor complex. G(s) is heterotrimeric (alpha beta gam ma) and activation can be accomplished by dissociation of the alpha-su bunit (G(s) alpha) from the beta gamma-subunit complex (G beta gamma). G(s) alpha is also a substrate for choleragen catalyzed ADP-ribosylat ion when it is associated with G beta gamma but not as free G(s) alpha . Using recombinant DNA techniques we modified the cDNA for the 52,000 M(r) form of Gsa (G(s) alpha(52)) to produce a protein with a 2,400 M (r) N-terminal extension (G(s) alpha(54.4)). This N-terminal extension could be removed with the protease Factor Xa. In vitro transcription and translation of the recombinant plasmid containing the cDNA's for G (s) alpha(52) and G(s) alpha(54.4) produced a 52,000 M(r) and a 54,000 M(r) protein, respectively. In solution the properties of G(s) alpha( 52) and G(s) alpha(51.4) were indistinguishable. Both proteins: (a) fo rmed a heterotrimer with G beta gamma and their affinities for the sub unit complex were the same; (b) could be ADP-ribosylated by choleragen in the presence but not in the absence of G beta gamma; (c) bound the non-hydrolyzable GTP analogue, GTP gamma S, and were protected from c hymotryptic proteolysis by the guanine nucleotide; and (d) could activ ate in vitro translated type IV adenylylcyclase. G(s) alpha(54.4) and G(s) alpha(52) were incorporated into S49 cyc(-) membranes, which lack Gsa. After incorporation, both G(s) alpha(52) and G(s) alpha(54.4) we re protected from chymotryptic proteolysis when GTP gamma S was presen t, revealing that both proteins were able to bind the nucleotide and u ndergo a conformational change characteristic of Gsa activation. When G(s) alpha(52) was incorporated into cyc(-) membranes it could mediate both hormone and GTP gamma S stimulation of adenylylcyclase and could be ADP-ribosylated by choleragen, but Gs alpha(54.4) could do neither of these things, indicating that the properties of G(s) alpha(54.4) w ere altered by the membrane. Deletion of the N-terminal extension by t reatment with Factor Xa in solution converted G(s) alpha(54.4) to G(s) alpha(52), and upon incorporation into cyc(-) membranes it behaved li ke G(s) alpha(52) in every regard, showing that the effect of the N-te rminal extension was reversible. A lack of other differences in the fu nctional properties of G(s) alpha(52) and G(s) alpha(54.4) suggests a correlation between the interaction of G(s) alpha with G beta gamma an d its ability to activate adenylylcyclase.